In this procedure, mitochondria is separated by differential centrifugation
from the bulk of nuclei, plastids, and cellular debris which differ
in particle size or density. Mitochondria are then further separated
from the remaining plastids and nuclear debris by gradient centrifugation.
Homogenization media for mitochondrial isolation contain an osmoticum,
EDTA, bovine serum albumin (BSA), 2-mercaptoethanol, and polyvinylpyrrolidone
(PVP) which binds phenolics.
Yield of mtDNA; 5-10 ug/ 100 g Green Fruits
MATERIALS AND SOLUTIONS
Fruit Grinding Buffer (1 liter)
0.3 M mannitol --------------------------------- 54.7 g
50 mM Tris-HCl (pH 8.0) ---------------------- 50 ml of 1 M Tris-HCl
10 mM EGTA ---------------------------------- 20 ml of 0.5 M EGTA
5 mM EDTA ----------------------------------- 10 ml of 0.5 M EDTA
0.2% BSA -------------------------------------- 2 g
1.0% PVP -------------------------------------- 10 g
50 mM 2-mercaptoethanol ---------------------- 3.5 ml of 14.4
M 2-mercaptoethanol
Deionized H2O to make a final volume of ------ 1 liter
DNase Digestion Buffer (1 liter)
0.3 M mannitol --------------------------------- 54.7 g
50 mM Tris-HCl (pH 8.0) ---------------------- 50 ml of 1 M Tris-HCl
10 mM EGTA ---------------------------------- 20 ml of 0.5 M EGTA
5 mM EDTA ----------------------------------- 10 ml of 0.5 M EDTA
0.2% BSA -------------------------------------- 2 g
50 mM MgCl2 ---------------------------------- 50 ml of 1 M MgCl2
Deionized H2O to make a final volume of ------ 1 liter
Gradient Buffer (1 liter)
0.3 M Sucrose ------------------------------------ 102.7 g
50 mM Tris-HCl (pH 8.0) ------------------------ 50 ml of 1 M Tris-HCl
20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
0.1% BSA ---------------------------------------- 1 g
Deionized H2O to make a final volume of -------- 1 liter
1.6 M Sucrose Step Gradient Buffer (100 ml)
1.6 M Sucrose ------------------------------------ 54.8 g
50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
0.1% BSA ---------------------------------------- 0.1 g
Deionized H2O to make a final volume of -------- 100 ml
1.2 M Sucrose Step Gradient Buffer (100 ml)
1.2 M Sucrose ------------------------------------ 41.1 g
50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
0.1% BSA ---------------------------------------- 0.1 g
Deionized H2O to make a final volume of -------- 100 ml
0.6 M Sucrose Step Gradient Buffer (100 ml)
0.6 M Sucrose ------------------------------------ 20.6 g
50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
0.1% BSA ---------------------------------------- 0.1 g
Deionized H2O to make a final volume of -------- 100 ml
ET buffer (100 ml)
20 mM EDTA ------------------------------------- 4 ml of 0.5 M EDTA
50 mM Tris-HCl (pH 8.0) ------------------------- 5 ml of 1 M Tris-HCl
Deionized H2O ------------------------------------ 91 ml
PROCEDURES
All steps should be performed in ice!
Ioslation of Mitochondria from Green Fruits
1. Collect young green fruits which are a reliable source of mtDNA.
Mitochondrial number may become smaller during ripening, since
mitochondrial protein, expressed as a percentage of cytoplasmic
protein, decreases during fruit maturations. In ripe fruit, compounds
produced also damage mitochondria during isolation.
2. Wash green fruits with tap water and chop into small pieces.
3. Add 2 volumes of Fruit Grinding Buffer per volume of fruit.
4. Grind 1,500 g of green fruits in a waring blender briefly.
5. Filter the homogenate through two layers of cheesecloth and
one layer of Miractoth.
7. Centrifuge the homogenate for 15 minutes at 1,500g and 4oC.
8. Filter the supernatant through one layer of Miracloth.
9. Harvest the crude mitochondria by centrifuging for 15 minutes
at 13,000g and 4oC.
10. Resuspend the pellet gently with a paint brush in 12 ml of
DNase Digestion Buffer.
11. Digest nuclear DNA with 120 ul DNase I (10 mg/ml) on ice for 45 minutes.
12. End the DNase treatment by adding 4 ml 0.5 M EDTA.
13. Dilute the preparation with 400 ml of Gradient Buffer.
14. Collect the mitochondria by centrifuging for 15 minutes at
13,000g and 4oC.
15. Resuspend mitochondrial pellet in 12 ml of Gradient Buffer.
16. Layer 3-4 ml of mitochondrial suspension per sucrose step
gradient consisting of 10 ml 1.6 M Sucrose Step Gradient Buffer,
10 ml 1.2 M Sucrose Step Gradient Buffer, and 10 ml 0.6 M Sucrose
Step Gradient Buffer.
17. Centrifuge for 1 hour at 25,000 rpm for purification from
contaminating subcellular structures and residual DNase.
18. After centrifugation, the bands at the interface will appear
green because of thylakoid contamination. Collect the lower band.
19. Dilute the mitochondrial fraction with 3 volumes of Gradient
Buffer slowly over a 15-minute period to minimize disruption by
osmotic shock.
20. Harvest the mitochondria by centrifugation at 15,000g for
10 minutes at 4oC.
21. Resuspend the mitochondrial pellet in 7 ml of ET buffer.
22. Add 350 ul of 10% Sarkosyl to lyse the mitochondria.
Optional; add 1 ul of Proteinase K (20 mg/ml) and incubate for 15 minutes on ice to reduce clumps
of debris.
CsCl Gradients for mtDNA Purification
23. To 7 ml of lysed mitochondria, add 8.05 g CsCl and 220 ul
of ethidium bromide (10 mg/ml).
24. Add ET buffer to make the total weight to 8.32 g. Dissolve
the CsCl.
25. Transfer the liquid to a Beckman Quick-Seal tube and heat-seal
the tube.
26. Centrifuge at 65,000 rpm for 8-10 hours at 20oC.
27. Collect both linear and supercoiled DNA bands with a syringe.
28. Extract the ethidium bromide three times with isopropanol
equilibrated with CsCl-saturated TE buffer.
29. Dialyze mtDNA against 4 liters of STE buffer for 1-2 hour.
30. Add 1/10th volume of 3 M Sodium acetate (pH 7.0) and 2.5 volume of ethanol.
31. Precipitate DNA at -20oC overnight.
32. Collect DNA by centrifugation at 15,000 rpm at 4oC for 20
minutes.
33. Resuspend mtDNA in 100-500 ul TE and store at -20oC or 4oC.
NOTES
Deoxyribonuclease (DNase) is used to remove remaining DNA external
to the mitochondria. The effectiveness of this DNase step requires
penetration of the DNase into nonintact contaminating plastids
and nuclear debris and sufficient mitochondrial integrity to prevent
the enzyme from entering the organelles. Integrity of mitochondria
is critical when utilizing a DNase step during purification. This
treatment should be performed early in the isolation procedure
while the highest proportion of mitochondria are intact.
In some mtDNA preparations, faint bands which comigrate with purified
plastid DNA restriction fragments can be seen on ethidium bromide-stained
gels of restriction enzyme-digested mtDNA preparations. Whether
these represent plastid DNA contamination can be checked by hybridizing
total plastid DNA (nick-translated) to a Southern blot.
If DNase treatment of sucrose gradient-purified fractions does
not give adequate purification from plastid DNA, dyes which enhance
separation of organelle DNAs can be incorporated into the CsCl
gradients. Plastid DNA could be quantitatively separated from
mtDNA on CsCl gradients containing diamidinophenylindol or bisbenzimide.
KIT INFORMATION
REFERENCES
Boeshore, ML, Lifshitz, I, Hanson, MR, Izhar, S,
(1983) Mol. Gen. Genet. 190: 459.